Answer: To eliminate bacteria that failed to take in the plasmid.
Explanation:
Research shows that plasmid DNA is only taken in by small percentage of bacteria in a large population.
Therefore, the need of growing transformed bacteria in the presence of ampicillin is very important. The main reason of this process is to kill off or eliminate bacteria that have failed to take in the plasmid, due to their possession of recombinant DNA plasmid pARA-R (an ampicillin resistance gene), that allows them to survive.
However, after completion of the aforementioned procedure, the bacteria that have been transformed will be able to take in the plasmid.
Answer:
- Bacteria cannot remove intronic sequence from a gene, so if the gene for factor VIII were transcribed, it would translate to a nonfunctional protein.
- The bacteria will not recognize the eukaryotic promoter to transcribe the gene.
Genome engineering steps:
I. Isolate by PCR the mature mRNA of the target factor VIII gene
II. Synthesize a complementary DNA (cDNA) for this mRNA by using a reverse transcriptase enzyme
III. Insertion of the cDNA into a plasmid vector
IV. Expression of the protein in the host bacterial cell through transformation
Explanation:
Bacteria lack the spliceosome (ie., the eukaryotic machinery required to remove introns from the primary mRNA), because bacterial genes do not have introns. Moreover, eukaryotic organisms require several proteins that bind to the promoter sequence and subsequently recruit the RNA polymerase to initiate this process. In bacteria, transcription is controlled by proteins that bind to cis-acting sequences which regulate the transcription of adjacent genes. In contrast to bacteria, eukaryotic genes have many classes of promoter and enhancer elements.
Genome engineering technologies can be used to produce bacteria expressing human proteins derived from synthetically introduced human genes (see steps above). For example, recombinant bacteria expressing the human insulin gene have already been designed by using E. coli as cell factories.
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